819
J Physiol 584.3 (2007) pp 819–834
Differences in sympathetic neuroeffector transmission to
rat mesenteric arteries and veins as probed by in vitro
continuous amperometry and video imaging
Jinwoo Park1 , James J. Galligan2 , Gregory D. Fink2 and Greg M. Swain1
1
2
Department of Chemistry and the Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA
Department of Pharmacology and Toxicology and the Neuroscience Program, Michigan State University, East Lansing, MI 48824, USA
As arteries are resistance blood vessels while veins perform a capacitance function, it might
be expected that sympathetic neural control of arteries and veins would differ. The function
of sympathetic nerves supplying mesenteric arteries (MA) and veins (MV) in rats was
investigated using in vitro continuous amperometry with a carbon fibre microelectrode
and video imaging. We simultaneously measured noradrenaline (NA) overflow at the blood
vessel adventitial surface and vasoconstriction evoked by electrical stimulation of perivascular
sympathetic nerves. Sympathetic nerve arrangement was studied using glyoxylic acid-induced
fluorescence of NA. We found that: (i) there were significant differences between MA and MV
in the arrangement of sympathetic nerves; (ii) frequency–response curves for NA overflow
and vasoconstriction for MV were left-shifted compared to MA; (iii) the P2X receptor
antagonist, pyridoxal-phosphate-6-azophenyl-2 ,4 -disulphonic acid (PPADS, 10 μM), reduced
constrictions in MA but not in MV while the α1 -adrenergic receptor antagonist, prazosin
(0.1 μM), blocked constrictions in MV but not in MA; (iv) NA overflow for MA was enhanced by
the α2 -adrenergic receptor antagonist, yohimbine (1.0 μM), and attenuated by the α2 -adrenergic
receptor agonist, UK 14,304 (1.0 μM), while yohimbine and UK 14,304 had little effect in MV;
(v) cocaine (10 μM) produced larger increases in NA overflow in MA than in MV; (vi) UK 14,304
constricted MV but not MA while yohimbine reduced constrictions in MV but not MA. We
conclude that there are fundamental differences in sympathetic neuroeffector mechanisms in
MA and MV, which are likely to contribute to their different haemodynamic functions.
(Received 11 April 2007; accepted after revision 29 August 2007; first published online 30 August 2007)
Corresponding author G. M. Swain: Department of Chemistry and the Neuroscience Program, Michigan State
University, East Lansing, MI 48824, USA. Email: [email protected]
Sympathetic nerve activity plays a prominent role in
regulating the resistance and capacitance functions of
arteries and veins, respectively. Arterial blood vessels
distribute oxygenated blood to tissues with diverse
functions and rapidly changing oxygen requirements.
Therefore, arterial directed sympathetic activity must
allow precise moment-to-moment control of regional
blood flow and systemic arterial pressure (Segal, 2000).
On the other hand, veins function as a blood reservoir.
Shifting blood from systemic veins to arteries in response
to orthostatic or other physiological disturbances requires
a rapid and sustained sympathetic activation of venous
smooth muscle (Tani et al. 2000). Highly innervated veins
of the splanchnic circulation are particularly important
contributors to this redistributive function of the venous
system (Daugirdas, 2001; Kreulen, 2003a). The nervous
system may generate the required differences in control
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
dynamics using differentiated patterns and/or levels of
nerve firing to arteries and veins. Differential control may
also occur at the neuroeffector junction. In fact, veins
differ significantly from arteries in their contractile and
electrical responses to sympathetic nerve stimulation or
to drugs that mimic the activity of sympathetic nerves
(Kreulen, 1986; Hottenstein & Kreulen, 1987; Bobalova
& Mutafova-Yambolieva, 2001). One reason for these
differences is that arteries and veins may be innervated
by distinct sympathetic neurons and these neurons
may communicate using different neurotransmitters.
Adenosine 5 -triphosphate (ATP) and noradrenaline (NA)
are neurotransmitters released by sympathetic nerves
supplying small (∼250 μm) mesenteric arteries while NA
is the primary vasoconstrictor released by perivenous
nerves (Hottenstein & Kreulen, 1987; Hirst & Jobling,
1989; Stjarne et al. 1994; Westfall et al. 1996).
DOI: 10.1113/jphysiol.2007.134338
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J. Park and others
Sympathetic neuroeffector transmission in arteries and
veins may differ because there are distinctive mechanisms
regulating neurotransmitter release and clearance.
Continuous amperometry with a microelectrode
has been used to examine neurotransmission in the
peripheral nervous system. With this method, movement
of an electroactive neurotransmitter from local neuroeffector junctions is measured as an oxidation current
that is proportional to the concentration at the blood
vessel surface. This technique provides time-dependent
information on the local concentration of a redox-active
molecule at the blood vessel surface that results from
release at nearby varicosities. The measured current
corresponds to efflux from multiple release sites
nearby the microlectrode and not release from a single
neuroeffector junction. For sympathetic nerves, NA is
the electroactive neurotransmitter monitored. Electrochemical methods have been used to study NA release
from sympathetic nerve endings in rat tail and mesenteric
arteries (Mermet et al. 1990; Gonon et al. 1993; Gonon,
1995; Dunn et al. 1999; Dugast et al. 2002; Brock & Tan,
2004; Teschemacher, 2005; Yavich et al. 2005). NA overflow
from perivascular nerves can be oxidatively detected in
the presence of other cotransmitters, including ATP and
neuropeptide Y (NPY), because the latter two compounds
are electroinactive. Therefore, electrochemical methods
are quite useful for mechanistic studies of endogenous
NA release and clearance in vascular tissues, but have not
yet been applied to the study of veins.
In the present work, continuous amperometry was used
to record NA overflow in vitro at the adventitial surface
of isolated rat mesenteric artery (MA) and vein (MV)
preparations in response to focal electrical stimulation.
Using a carbon fibre microelectrode, oxidation current
measurements were made that provided information
regarding the temporal change in NA concentration
at the blood vessel surface in response to a stimulus.
This concentration is determined by complex and highly
regulated mechanisms effecting both transmitter release
and clearance. Pharmacological methods were used to
investigate some of these mechanisms. Video imaging was
used to monitor blood vessel constriction simultaneously
with changes in the NA oxidation current. The combined
measurement of NA and vessel diameter provides detailed
information on the time-dependent relationship between
vascular contraction and NA concentration at the blood
vessel surface. We used these techniques to test the
hypothesis that there are fundamental differences in the
dynamics of sympathetic neuroeffector transmission in
mesenteric arteries and veins.
Methods
Carbon fibre microelectrode preparation
Heat-treated (3000◦ C) pitch-base type carbon fibres
(formerly AVCO Specialty Materials, Inc., Lowell, MA,
J Physiol 584.3
USA) having a nominal diameter of 35 μm were used.
Each carbon fibre was attached to a longer copper wire
with conductive silver epoxy. The carbon fibre–copper
wire assembly was then inserted into a polypropylene
pipette tip and the tapered end was carefully heated in
a micropipette puller. This softened the polypropylene
and caused it to flow over the carbon fibre surface, thus
insulating the electrode (Chow et al. 1992; Zhou & Misler,
1996; Park et al. 2005). The resulting microelectrode
possessed a cylindrical architecture with an exposed length
of ca 500 μm (geometric area = 5 × 10−4 cm2 ). Electrodes
insulated with polypropylene are generally non-toxic to
tissue and chemically resistant to the isopropyl alcohol
(IPA) used for electrode cleaning (Ranganathan et al.
1999). The carbon fibre was used in vitro without any
electrochemical pretreatment for activation or Nafion
coating for fouling protection (Stamford, 1986). We
previously reported that a 25% response (i.e. sensitivity)
attenuation in the measured oxidation current for NA
is typical for a bare carbon fibre during the first 2 h
of exposure to a mesenteric blood vessel, if most of
the adipose and connective tissue is first removed. Little
change in the response occurs thereafter (Park et al. 2005).
In the present work, a new carbon fibre was exposed
to the tissue and the flowing physiological buffer for a
period of about 2 h in order to equilibrate the tissue and
to stabilize the response prior to the start of a series of
measurements. Recent work by our group has shown that
diamond microelectrode offers advantages over traditional
carbon fibre for in vitro electrochemical measurements of
NA in terms of response sensitivity, reproducibility and
stability (Park et al. 2005, 2006a,b). However, we were
unable to make use of this microelectrode in this work
because some of the drugs employed were oxidatively
detected at the positive potential used to measure NA. This
was not the case for carbon fibre as NA was detected at a
less positive potential, a value at which the drugs were
non-responsive.
Preparation of mesenteric vessels for in vitro
electrochemical and contractile measurements
The Institutional Animal Care and Use Committee at
Michigan State University approved all animal handling
and use procedures. The male Sprague–Dawley rats
(380–430 g, Charles River Inc, Portage, MI, USA) used
in these studies were killed with a lethal pentobarbital injection (50 mg kg−1 , i.p.). The abdomen was
then opened and the small intestine was carefully removed
and placed in an oxygenated (95% O2 , 5% CO2 ) Krebs
buffer solution of the following composition: 117 mm
NaCl, 4.7 mm KCl, 2.5 mm CaCl2 , 1.2 mm MgCl2 , 25 mm
NaHCO3 , 1.2 mm NaH2 PO4 , and 11 mm glucose. A section
of the mesentery close to the ileal wall was carefully
cut free from the intestine and transferred to a small
silicone elastomer-lined (Sylgard 184, Dow Corning)
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
J Physiol 584.3
Neuroeffector transmission in arteries and veins
Teflon bath (4.8 ml volume) where it was gently stretched
and pinned flat. Secondary or tertiary arteries and veins
(180–330 μm outer diameter) were then isolated for in
vitro study by carefully cutting away the surrounding
adipose and connective tissues under a dissecting
microscope. The bath was mounted on the stage of
an inverted microscope (Olympus CX41, USA) and
superfused continuously with 37◦ C Krebs solution, pH 7.4,
at a flow rate of 5–6 ml min−1 . The solution flow was
controlled by a peristaltic pump (Masterflex, Cole Parmer,
USA).
Prior to use, the carbon fibre microelectrode was
cleaned by soaking in distilled isopropyl alcohol (IPA)
for at least 15 min (Ranganathan et al. 1999). The
microelectrode was then affixed to a micromanipulator
(MP-1, Narishige Instruments, Japan), which enabled
its reproducible positioning against the side of a MA
or MV. The cylindrical portion of the microelectrode
was positioned alongside the vessel so that the NA flux
from nearby release sites was sensed by most of the
electrode surface. This positioning allowed the microelectrode to move in conjunction with the blood vessel
as it contracted and relaxed, thus enabling measurement
of NA at the blood vessel surface with an unchanging
electrode-blood vessel distance. We confirmed this by
measuring the NA oxidation current elicited by electrical
stimulation in the presence and absence of the P2X
antagonist, PPADS, and the α 1 antagonist, prazosin, which
together abolished arterial constriction. The oxidation
currents measured were the same with and without the
drugs. The microelectrode and tissue preparation were
positioned in the centre of the bath, equidistant from the
inlet and outlet solution ports. A platinum wire counter
and a commercial ‘no leak’ Ag–AgCl (3 m KCl, model
EE009, Cypress Systems Inc., USA) reference electrode
were also mounted in the bath to complete the electrochemical cell. All electrochemical measurements were
made with an Omni 90 analog potentiostat (Cypress
Systems Inc.), an analog-to-digital converter (Labmaster
125) and a computer running Axotape software (version
2.0, Axon Instruments, Union City, CA, USA). Continuous
amperometric i–t curves were recorded at a detection
potential of 400 mV. At this potential, NA is oxidized at a
mass-transfer limited rate. The analog voltage output from
the potentiostat, a value that reflects the current flowing
through the working electrode, was low-pass filtered at a
time constant of 200 ms (5 Hz) before being digitized using
an A/D converter at a sampling rate of 100 Hz. The data
were then stored on a computer for further processing. All
measurements were made in unpressurized blood vessels.
Focal stimulation of perivascular nerves
Short trains of electrical stimulation were used to
trigger NA release. Perivascular nerves were stimulated
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
821
using a bipolar focal stimulating electrode positioned
along the surface of the blood vessel at a distance
of ca 200 μm from the carbon fibre. This positioning
minimized stimulus artifact in the current recordings. The
focal stimulating electrode consisted of two AgCl-coated
Ag wires inserted into a double-barrelled capillary glass
(tip diameter = 180 μm). The wires were connected to a
stimulus isolation unit and a Grass Instruments stimulator
(S88, Quincy, MA, USA). Trains of 60 stimuli (0.3 ms
pulse width, 30–70 V) at frequencies ranging between
0.5 and 20 Hz were used (Park et al. 2006a,b) to evoke
neurotransmitter release.
Video monitoring of vasoconstriction
The output of a black–white video CCD camera (Hitachi
KP-111) attached to the inverted microscope was fed to
a frame grabber card (Picolo, Euresys Inc., TX, USA)
mounted in a personal computer. Video images were
analysed real time using Diamtrak edge-tracking software
(version 3.5, http://www.diamtrak.com), which tracks the
distance between the outer edges of the blood vessel in
the observation field. Changes in blood vessel diameter
of 1 μm could be resolved. The digitized signal was
converted to an analog signal (DAC-02 board, Keithley
Metrabyte, Tauton, MA, USA) for subsequent processing
by an analog-to-digital converter (Labmaster 125) and
analysis in a second computer running Axotape for a
permanent recording of blood vessel diameter as function
of time. Analog signals were sampled at 100 Hz and data
were stored on the computer’s hard drive for subsequent
analysis and display.
Drug application
All chemicals and drugs were reagent-grade quality, or
better, and were used without additional purification.
Ultrapure water (distilled, deionized, and filtered over
activated carbon, 17–18 M, Barnstead E-Pure System)
was used for all solution preparation and cleaning. Drugs
were added to the superfusing Krebs solution; it took
ca 2 min for them to reach the tissue and they were
applied for 20 min prior to assessing their effects on
nerve-mediated responses. Drugs were obtained from
Sigma-Aldrich Chemical Company (St Louis, MO, USA).
Glyoxylic acid fluorescence histochemistry
Second- or third-order veins and arteries from the ileal
mesentery were isolated by carefully cutting away the
surrounding adipose and connective tissues. Red blood
cells were flushed out of the blood vessel lumen with
phosphate buffer (0.1 m, pH 7.2) using a 30-ga hypodermic
needle and syringe, and tissues were cut to 1 cm lengths.
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J. Park and others
For these experiments, we used 20 MA and MV segments
from five rats. Catecholamine fluorescence was revealed
after incubating tissues in a 2% glyoxylic acid–0.2 m
phosphate buffer (pH 7.0) solution for 5 min at room
temperature. The blood vessels were then stretched onto
glass slides and heated at 80◦ C for 5 min. Preparations were
mounted in mineral oil and observed using a fluorescence
microscope (Nikon Eclipse TE 2000-U) equipped with
a filter set, UV2E/C (excitation filter, 340–380 nm and
emission, 435–485 nm).
Data analysis
Analysis of the current (i)–t and diameter–t profiles
was performed using Clampfit 8.1 as part of the
pCLAMP 8.1 software package (Axon Instruments). Mean
values were compared by using Student’s paired t test
(GraphPad Software, San Diego, CA, USA). P < 0.05 was
regarded as statistically significant. Data are reported as
mean ± standard error of the mean (s.e.m.) with ‘n’ values
indicating the number of rats from which the data were
obtained. MA and MV constrictions are expressed as a
percentage of the initial resting diameter (in μm) of the
J Physiol 584.3
blood vessel. For nerve stimulation frequency–response
curves, the half-maximal effective stimulation frequency
(S50 ) and maximum constriction (E max ) were calculated
from curves obtained in individual tissues using the
following function:
Y =E max X /(S50 +X )
Where X is the stimulation frequency tested and Y is the
peak response amplitude.
Results
Distribution of sympathetic nerve fibres associated
with MA and MV
Glyoxylic acid (GA) reacts with NA to form an intensely
fluorescent
2-carboxymethyl-dihydroisoquinoline
derivative (Bjorklund et al. 1972). The distribution
of GA-induced fluorescence of neuronal stores of NA
was used to assess the disposition of periarterial and
perivenous sympathetic nerves. GA-induced fluorescence
revealed a network of varicose nerve fibres around MA
and MV (Fig. 1A and B). The periarterial nerve plexus
Figure 1. Glyoxylic acid-induced fluorescence of catecholamines in perivascular sympathetic nerves
A and B show low magnification (100×) images of sympathetic nerves while C and D show high magnification
images (400×). The nerve plexus in arteries has a mesh-like arrangement of nerve fibres that are orientated along
both the longitudinal and circular axis of the blood vessel (C). The plexus in veins has a selective circular arrangement
with very few fibres orientated along the longitudinal axis of the blood vessel (D).
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
J Physiol 584.3
Neuroeffector transmission in arteries and veins
consisted of bundles of several axons arranged in a
net-like manner. The perivenous plexus was less dense
than in arteries and consisted of individual varicose axons
largely orientated circumferentially around the vessel.
Higher magnification images clearly reveal the differential
nerve arrangement (Figs 1C and D). The difference
in periarterial and perivenous sympathetic nerve
arrangement was quantified by counting the number
of nerve fibres crossing horizontal (longitudinal axis)
and vertical (radial axis) grids superimposed on a
vessel image (400× magnification). The number of
vertical and horizontal fibre crossings was similar in
MA (vertical = 9.7 ± 1.2, horizontal = 9.6 ± 1.2, n = 5),
whereas there were fewer horizontal than vertical crossings
in MV (horizontal = 3.6 ± 0.4, vertical = 6.8 ± 0.9, n = 5;
P < 0.05). In addition, the number of vertical crossings in
MV was significantly less than in MA (P < 0.05).
NA oxidation currents and constriction in MA and MV
The oxidation current measured at 400 mV reflects the
concentration of endogenous NA at the blood vessel
surface in the vicinity of the recording microelectrode
(Park et al. 2005, 2006a,b). This time-dependent
concentration is the net result of release from multiple
varicosities near the microelectrode, clearance by
neuronal reuptake, and diffusional and convective mass
transport away from the release sites, the latter due to the
fluid dynamics in the bath. Preparations were discarded
for which the electrically evoked oxidation current or
constriction were not blocked by tetrodotoxin (TTX,
0.3 μm). Several locations on a blood vessel were probed
for NA oxidation currents in order to find one that yielded a
‘large’ current (i.e. hot spot). Finding such a spot was more
difficult in MV and this is consistent with the differential
arrangement of sympathetic nerve fibres revealed in
Fig. 1. Sites yielding the largest oxidation current were
then selected for comparative studies of perivenous and
periarterial nerves. Measurable NA release was only evoked
by trains of electrical stimuli (see methods section) and not
by a single pulse stimulation.
The same trains of electrical stimuli produced
frequency-dependent constrictions of MA and MV with
NA oxidation currents and constriction being detected
at a lower threshold frequency in MV (0.5 and 0.2 Hz,
respectively) than in MA (1 and 0.5 Hz, respectively).
Figure 2A and B displays representative recordings of
constriction (top) and the NA oxidation current (bottom)
for MA and MV at stimulation frequencies of 3 and 20 Hz.
It can be seen that at 20 Hz, both vessels constrict by
Figure 2. In vitro continuous amperometric measurement of NA oxidation currents and video imaging
of neurogenic constriction in a MV
NA oxidation currents and neurogenic constrictions are frequency dependent. Neurogenic constrictions (top)
and NA oxidation currents (bottom) caused by 3 and 20 Hz stimulus trains (60 pulses with a 0.3 ms pulse
width) in a MA (A) and a MV (B). The bars under the current traces denote the period of nerve stimulation.
C and D, frequency–response curves for NA-oxidation current (C) (∗ significantly different from MA, P < 0.05) and
constriction in MA and MV (D). Data are means ± S.E.M.
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
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J. Park and others
ca 25%. The vessel constricts rapidly in response to the
stimulus followed by a slow relaxation back to the resting
diameter. However, 3 Hz was the threshold stimulation
for a detectable arterial constriction while this frequency
evoked a significant constriction of veins. At 20 Hz, the
peak oxidation currents in MA and MV were 12.7 and
23.8 pA, respectively. At 3 Hz, peak currents in MA and
MV were 5.1 and 9.8 pA, respectively.
Figure 2C and D contains summary plots of the
oxidation currents and constrictions for MA and MV.
The data demonstrate a frequency-dependent increase
in the NA oxidation current and constriction for MA
and MV. These data also reveal that NA oxidation
currents were greater for MV than for MA and that the
frequency–response curve for nerve stimulation-induced
constriction in MV was left-shifted from the curve for
MA. The half-maximum stimulation frequency (S50 ) of
the constriction was 1.9 ± 0.2 Hz for MV, and this value
was significantly lower (P < 0.05) than the 4.8 ± 0.3 Hz for
MA. The maximum constriction (E max ) was similar in MV
(28 ± 2%) and MA (31 ± 3%).
Influence of PPADS and prazosin on NA oxidation
current and vasoconstriction
Along with NA acting on α 1 -adrenergic receptors,
ATP acting at P2X purinergic receptors contributes to
sympathetic neurogenic constriction of arteries in the rat
mesentery (Dunn et al. 1999; Gitterman & Evans, 2001;
Luo et al. 2003). In order to study the noradrenergic
component of the constriction independently, the P2X
receptor antagonist, PPADS (10 μm), was used to
block the constricting effect of ATP. Figure 3A shows
that PPADS did not alter the NA oxidation current
elicited by 20 Hz stimulation but did substantially
reduce the MA constriction. In contrast, PPADS affected
neither the oxidation current nor constriction of MV
(Fig. 3B). This result indicates that ATP largely mediates
neurogenic constriction of MA while NA mediates
neurogenic constriction of MV.
The α 1 -adrenergic receptor antagonist, prazosin
(0.1 μm), was also applied to the tissues individually and
in combination with PPADS. Prazosin reduced the neurogenically mediated constriction of MA while PPADS and
prazosin together totally blocked constriction without
altering the NA oxidation current. In contrast, PPADS had
no effect on either constriction or NA oxidation current
from MV, while prazosin blocked constriction (Fig. 3D)
but had no effect on NA oxidation current.
Quantitative determination of NA at the blood
vessel surface
Determining the proportionality between the measured
current and the local NA concentration is a complex
J Physiol 584.3
issue. It is supposed that much of the electrode surface
is sensing the NA overflow, so we tried to mimic this type
of release by calibrating the microelectrode in the flow
bath using different injected concentrations of NA. This
was accomplished by introducing a NA solution through
a non-metallic (combination of plastic and fused silica)
needle (i.d. 250 μm, MicroFil) that was positioned at a
fixed distance from the microelectrode (ca 10 μm, Park
et al. 2005). Calibration was performed before and after
a series of measurements at the blood vessel to verify
the minimal response attenuation occurred. Oxidation
currents increased proportionally with the injected NA
concentration between 5 and 300 nm (r 2 > 0.995). The
minimum detectable concentration was 5–10 nm (signal to
noise ratio = 3). Based on this calibration, the maximum
oxidation current elicited by a 10 Hz stimulus train
corresponds to an apparent concentration between 10
and 30 nm. This value is in good agreement with values
(40–120 nm) reported for the densely innervated rat
artery elicited by a 5 Hz, 100 s stimulation (Mermet et al.
1990; Gonon et al. 1993). Comparison of the apparent
NA concentration–constriction curves in the presence of
PPADS for MA and MV indicates that MA are relatively
insensitive to endogenously released NA with a maximum
constriction of ca 7% at 20 nm (Fig. 3C). MV are more
sensitive to endogenous NA as 20 nm caused a 3-fold
greater constriction (Fig. 3D).
Kinetics of NA oxidation currents and neurogenic
constriction in MA and MV
The kinetics of NA release and clearance, and
vasoconstriction were quantitatively different in MA and
MV. Figure 4 shows superimposed temporal profiles for
MA and MV constriction (top) and NA oxidation current
(bottom) evoked by a 20 Hz train of stimuli. The rising
portion of the oxidation current reflects the kinetics of
release offset by the kinetics of clearance (reuptake and
mass transfer) while current decay reflects NA clearance
kinetics. The representative traces illustrate that the latency
for onset of constriction is shorter in MA but the rise time
of constriction is more rapid in MV. The decay time for the
constriction is longer in MV. There was no difference in
the latency to onset of the oxidation current between MA
and MV. The rise time of the current was shorter in MV
but the decay time in MV was longer than that in MA. The
rate of rise of the oxidation current elicited by 3 and 20 Hz
stimulation in MV was significantly faster than that in MA
(Fig. 5A). However, there were no artery–vein differences
in the decay half-time of the oxidation currents (Fig. 5B).
The rate of constriction (μm s−1 ) for MV was more rapid
than that for MA (Fig. 5B). The constriction duration was
longer for MV than MA at both stimulation frequencies
(Figs 5C and D).
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
Neuroeffector transmission in arteries and veins
J Physiol 584.3
A
B
Artery
Diameter
825
Vein
20 μm
20 μm
5s
5s
PPADS
PPADS
5 pA
Current
5 pA
5s
20 Hz
% Constriction
40
30
D
Control (n = 11)
Prazosin (n = 9)
PPADS (n = 4)
PPADS + Prazosin (n = 7)
40
% Constriction
C
5s
20 Hz
20
10
0
0 5 10 15 20 25 30 35
Apparent NA Concentration (nM)
Control (n = 6)
Prazosin (n = 4)
PPADS (n = 3)
30
20
10
0
0 5 10 15 20 25 30 35
Apparent NA Concentration (nM)
Figure 3. Contribution of α1 -adrenergic and P2X purinergic receptors to neurogenic constriction of MA
and MV
A, effects of PPADS (10 μM) on neurogenic constriction (top) and NA-oxidation current caused by a 20 Hz
stimulus train (60 pulses with a 0.3 ms pulse width) in a MA. PPADS blocked the constriction but not the NA
oxidation current. B, PPADS did not alter the neurogenic constriction or NA oxidation current in a MV. C, apparent
concentration–constriction curves for neurally released NA in MA in the absence and presence of PPADS and
prazosin (0.1 μM). Peak NA oxidation currents were measured during stimulus trains (0.2–20 Hz) by converting
oxidation currents to apparent NA concentrations using calibrated electrodes. The curve obtained in the presence
of PPADS (square symbols) reveals the sensitivity of MA to neurally relased NA. D, experiments similar to ‘C’ except
these studies were done in MV. The curve obtained in the presence of PPADS () reveals the sensitivity of MV to
neurally relased NA. Prazosin blocked the constriction caused by nerve stimulation in MV but it did not alter the
NA oxidation current (not shown). Data are means ± S.E.M.
Influence of prejunctional α2 -adrenergic receptors on
NA oxidation currents and vasoconstriction
Prejunctional α 2 -adrenergic autoreceptors located at the
sympathetic nerve terminals are activated by NA and
function to regulate its release (Langer, 1980; Stjarne
et al. 1994; Dunn et al. 1999; Westfall et al. 2002;
Brock & Tan, 2004). The influence of the α 2 -adrenergic
receptor antagonist, yohimbine (1.0 μm), and the agonist,
UK 14,304 (1.0 μm), on NA oxidation currents and
constriction of MA and MV was studied. MA constriction
at 20 Hz was unchanged by yohimbine (Fig. 6A) while MV
constriction decreased by 13%. (Fig. 6B). Apparently, α 2
receptors contribute to NA-induced constriction of MV.
Yohimbine increased the maximum oxidation current for
MA by 66% from 16.6 to 27.6 pA, whereas yohimbine
increased the current for MV by only 9% from 16.1 to
17.5 pA (Fig. 6C). With the α 2 -adrenergic autoreceptors
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
Latency
25 um
NS
slope
Vein
Artery
10 pA
5s
Figure 4. Kinetics of NA oxidation current and neurogenic
vasoconstriction in MA and MV
Responses were evoked using a 20 Hz stimulus train (60 pulses with a
0.3 ms pulse width). The dotted line shows the onset of nerve
stimulation.
826
J. Park and others
blocked, NA overflow for MA was greater than that for
MV at all frequencies (Fig. 6C). The largest increases
were observed in MA and MV at the lower stimulation
frequencies. Figure 6D shows that the elevated junctional
NA concentration in the presence of yohimbine increased
the contractile response for MA at frequencies up to
about 7 Hz, as compared to control. Above this frequency,
constriction was the same with and without the drug
presumably due to saturation of postjunctional receptors.
In contrast, MV constriction was unaltered by yohimbine
at frequencies up to 7 Hz, above which constriction
declined.
Tables 1 and 2 present a statistical analysis of the NA
oxidation current and contractile response data with and
without (control) yohimbine in MA and MV, respectively.
In the presence of yohimbine, the rate of rise (slope) of
the oxidation current increased significantly at both 3 and
20 Hz for MA, but there was no significant difference in
the half-decay time or the constriction rate. The rate of
rise of the current and the half-time of current decay
1.0
0.8
0.6
0.4
0.2
0.0
Half decay time (s)
C
*
Artery
Vein
4
*
3 Hz
2
1
20 Hz
30
*
Artery
Vein
25
20
15
*
10
5
0
D
Artery
Vein
3 Hz
35
20 Hz
3
0
B
Constriction rate (μm/s)
1.2
were increased significantly in the presence of yohimbine
only at 3 Hz for MV. The rate of constriction was reduced
by yohimbine at both stimulation frequencies while the
relaxation times were unchanged by yohimbine in MV
(Table 2).
Figure 7A and B shows constrictions and oxidation
currents for MA and MV in response to a 20 Hz train
of stimuli with and without (control) the α 2 receptor
agonist, UK 14,304. UK 14,304 decreased the oxidation
current in MA by 73% from 16.5 to 4.4 pA, whereas
the current for MV decreased by only 29% from 20.8 to
14.8 pA. Constrictions of MA decreased from 28 to 17%
in the presence of UK 14,304 while the response for MV
decreased from 28 to 13% at 20 Hz. UK 14,304 constricted
MV by 15 ± 6% (n = 6) in the absence of any electrical
stimulation, consistent with the presence of postjunctional
α 2 receptors in the smooth muscle cells of MV. The plots
in Fig. 7C show that oxidation currents were abolished in
MA by UK 14,304 except at high frequencies. On the other
hand, NA currents in MV were reduced less by UK 14,304.
Constriction decay (s)
Rate of rise (pA/s)
A
J Physiol 584.3
3 Hz
80
70
60
*
20 Hz
Artery
Vein
*
50
40
30
20
10
0
3 Hz
20 Hz
Figure 5. Comparison of the time course of NA oxidation currents and constrictions in MA and MV
A, rate of rise of the NA oxidation current is significantly faster in veins (n = 20) compared to arteries (n = 10).
B, the rate of constriction was faster in veins compared to arteries. C, half-decay time of the NA oxidation current
was not different in arteries and veins. Oxidation currents were evoked by 3 and 20 Hz trains of stimuli. D, the
time to decay of constriction in veins was significantly longer than that in arteries after both 3 and 20 Hz trains of
stimulation. ∗ Significantly different from value in arteries (P < 0.05).
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
Neuroeffector transmission in arteries and veins
J Physiol 584.3
Constrictions in MA and MV were significantly reduced
by UK 14,304 (Fig. 7D).
Influence of cocaine and yohimbine on NA oxidation
currents and vasoconstriction
NA is cleared from the neuroeffector junction by the
noradrenealine transporter (NET), which is blocked by
cocaine (Mermet et al. 1990; Gonon et al. 1993). Figure 8A
and B shows constrictions (top) and oxidation currents
(bottom) for MA and MV elicited by a 20 Hz train of
stimuli before (control) and after the addition of cocaine
(10 μm). Cocaine increased the maximum NA oxidation
current in MA by 38% from 11.6 to 15.9 pA, whereas the
current for MV was unchanged. There was a slight increase
A
in MA constriction in the presence of cocaine; however,
no change was seen in MV. The summary plots shown in
Fig. 8C and D reveal that cocaine increased the oxidation
current in MA at all frequencies. The largest increases
(> 150% of control) occurred at the lower stimulation
frequencies. MA constriction increased in the presence of
cocaine at frequencies up to 7 Hz, above which no change
was seen. Oxidation currents or neurogenic constriction
in MV were not altered significantly by cocaine at any
stimulation frequency (Fig. 8C and D). Tables 1 and 2
present a statistical analysis of the NA oxidation current
and contractile response data in MA and MV in the absence
and presence of cocaine. Cocaine increased the rate of
rise and the half-decay time of the oxidation current in
MA at 3 and 20 Hz stimulation. Cocaine also increased
B
Artery
Vein
Diameter
20 μm
5s
5 pA
5s
Current
20 Hz
20 Hz
C
D
40
Artery/Yohimbine (n = 6)
Artery (n = 5)
Vein (n = 4)
Artery/Yohimbine (n = 5)
Vein/Yohimbine (n = 4)
Vein/Yohimbine (n = 6)
300
#
#
200
#
#
#
#
100
0
1
3
10
20
7
Frequency (Hz)
% Constriction
NE Overflow (% control)
400
30
20
10
0
1
10
Frequency (Hz)
Figure 6. Effect of yohimbine on NA oxidation currents and neurogenic constriction of MA and MV
A, recordings of oxidation current and constriction in a MA in the absence and presence of yohimbine (1 μM). B,
oxidation current and constriction in a MV with and without yohimine. Yohimbine increased the current but not
constriction in MA. Yohimbine had little effect on the oxidation current in MV but it reduced the constriction. Bars
under the current traces indicate the period of nerve stimulation (60 pulses, 0.3 ms pulse duration.) C, percentage
increase in NA oxidation currents in MA and MV in the presence of yohimbine. D, frequency–response curves for
vasoconstriction in MA and MV in the absence (control) and in the presence of yohimbine. #Significantly different
from control (P < 0.05). Data are means ± S.E.M.
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
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J. Park and others
J Physiol 584.3
Table 1. Effects of yohimbine (1 μm), cocaine (10 μm) and cocaine with yohimbine on the time course of NA oxidation
currents and constriction of mesenteric arteries
Hz
Control
(n = 10)
Yohimbine
(n = 4)
Cocaine
(n = 4)
Cocaine + Yohimbine
(n = 4)
Current rate of rise (pA s−1 )
3
20
0.05 ± 0.01
0.79 ± 0.07
0.09 ± 0.01∗
1.33 ± 0.17∗
0.18 ± 0.05∗
1.28 ± 0.28∗
0.19 ± 0.05∗
1.89 ± 0.32∗
Current half decay (s)
3
20
2.85 ± 0.15
3.16 ± 0.34
4.52 ± 1.76
5.04 ± 1.17
6.93 ± 1.18∗
5.74 ± 1.97∗
9.81 ± 0.89∗
8.17 ± 1.52∗
Constriction rate (mm s−1 )
3
20
3.31 ± 0.92
21.27 ± 1.34
6.54 ± 1.23
21.57 ± 1.08
9.58 ± 0.98∗
20.94 ± 1.75
6.12 ± 0.87∗
19.53 ± 1.81
Relaxation time (s)
3
20
7.36 ± 0.43
13.95 ± 1.37
8.86 ± 3.29
12.1 ± 3.5
7.85 ± 2.47
20.71 ± 3.23∗
9.14 ± 2.81
9.63 ± 2.52
Data are mean ± S.E.M. and are reported for 3 and 20 Hz stimulations. ∗ Significant difference from control values
Table 2. Effects of yohimbine (1 μm), cocaine (10 μm) and cocaine with yohimbine on the time course of NA oxidation
currents and constriction of mesenteric veins
Hz
Control
(n = 20)
Yohimbine
(n = 4)
Cocaine
(n = 4)
Cocaine + Yohimbine
(n = 4)
Current rate of rise (pA s−1 )
3
20
0.09 ± 0.01
1.14 ± 0.08
0.15 ± 0.02∗
1.12 ± 0.12
0.24 ± 0.09∗
1.14 ± 0.32
0.14 ± 0.01∗
0.82 ± 0.16
Current half decay (s)
3
20
3.12 ± 0.29
2.70 ± 0.31
4.68 ± 0.59∗
3.64 ± 0.82
6.72 ± 1.54∗
2.47 ± 0.53
6.30 ± 0.77∗
4.67 ± 0.94
Constriction rate (μm s−1 )
3
20
12.67 ± 1.73
31.53 ± 2.91
3.01 ± 0.40∗
13.27 ± 3.13∗
18.16 ± 3.94
20.96 ± 5.17
4.43 ± 0.22∗
27.57 ± 4.09
Relaxation time (s)
3
20
53.20 ± 13.5
51.90 ± 9.80
38.7 ± 7.7
19.6 ± 2.9
116 ± 38
79.9 ± 36.4
39.54 ± 7.87
47.63 ± 9.59
Data are mean ± S.E.M. and are reported for 3 and 20 Hz stimulations. ∗ Significant difference from control values.
the constriction rate in MA for the 3 but not the 20 Hz
stimulation, while the constriction relaxation time was
prolonged only after the 20 Hz stimulation (Table 1).
Cocaine increased the rate of rise of the NA oxidation
current and prolonged the current half-decay time only at
3 Hz stimulation in MV. The time course of constriction
was unaltered by cocaine in MV (Table 2).
In order to study the interplay between reuptake and
the prejunctional α 2 -adrenergic autoreceptor, cocaine
(10 μm) and yohimbine (1 μm) were used together to
study the combined effect on NA oxidation currents and
constriction. Figure 9A and B shows constriction and
oxidation currents in response to a 3 Hz train of stimuli
in the absence (control) and presence of cocaine and
yohimbine. Combined drug treatment increased oxidation
currents in MA above the response elicited by either drug
alone (Figs 6A and 8A). An increase was also observed
for MV but not to the same level as MA. For example,
the NA oxidation current for MA increased by 656% in
the presence of both drugs (4–30 pA), whereas a smaller
increase of 333% from 5 to 15 pA was seen for MV. MA
constriction increased by 82% while the response for MV
increased by only 8% at 3 Hz. Figure 9C, D shows plots
of the oxidation currents and constriction (% of control)
at different stimulation frequencies. Larger increases in
oxidation current occurred in MA compared to MV
particularly at low stimulation frequencies. The contractile response increased at frequencies < 7 Hz but not at
high frequencies for MA, whereas the response for MV
was decreased at all frequencies. The data in Table 1 show
that the combined drug application significantly increased
all values related to oxidation current in MA at both 3
and 20 Hz. Combined drug treatment had only a modest
effect on constriction in MA (Table 1). The rate of rise of
the oxidation current, current half-decay and constriction
rate were increased significantly only at 3 Hz in MV in the
presence of both drugs (Table 2).
Discussion
Arrangement of nerves
MA and MV are supplied by sympathetic nerves at the
medial–adventitial border, but there are fewer nerve fibres
in MV than in MA (Furness & Marshall, 1974; Nilsson
et al. 1986). This does not necessarily indicate that the
innervation density of MV is less than MA. MV have a
thinner medial layer than MA so the number of axons per
smooth muscle cell could be similar in MA and MV. This
supposition is supported by the fact that ultrastructural
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
Neuroeffector transmission in arteries and veins
J Physiol 584.3
studies have shown that guinea pig MA and MV possess
a similar density of neuromuscular junctions (Klemm
et al. 1993). The nerve plexus for MA consists of bundles
of axons arranged in a mesh-like network with nerve
fibres equally likely to run parallel or perpendicular to
the longitudinal axis of the vessel. For MV, the plexus
consists of single axons with a circumferential nerve fibre
arrangement. This arrangement made optimal detection
of NA oxidation current dependent on the position of
the recording microelectrode, more so than for MA.
Small microelectrode movements (≤ 50 μm) resulted in a
marked change in NA oxidation current for MV. Therefore,
NA oxidation current measurements were made with MV
A
only after the electrode had been optimally positioned to
record the maximum current.
In vitro continuous amperometry
Our data, and results published by other groups (Gonon
et al. 1993; Dunn et al. 1999; Brock et al. 2000), show
that NA at the blood vessel surface, caused by release from
perivascular sympathetic nerve fibres, can be measured
using in vitro continuous amperometry. The electrochemical method is particularly useful for investigating
NA dynamics in real time near the sites of release.
Amperometry with a microelectrode provides different
B
Artery
829
Vein
Diameter
20 μm
5s
5 pA
5s
Current
20 Hz
20 Hz
C
300
Artery/UK 14,304 (n = 4)
250
Vein/UK 14,304 (n = 5)
200
150
100
50
0
*
1
*#
*#
#*
*
*#
*
*
Artery (n = 5)
Vein (n = 5)
Artery/UK 14,304 (n = 5)
Vein/UK 14,304 (n = 5)
40
*#
*
10
20
3
7
Frequency (Hz)
% Constriction
NE Overflow (% control)
D
30
20
*
10
*
*
*
0
1
10
Frequency (Hz)
Figure 7. Effect of UK 14,304 on NA oxidation currents and neurogenic constriction of MA and MV
A, recordings of constriction (upper traces) and oxidation currents (lower traces) in a MA in the absence and presence
of UK 14,304 (1 μM). UK 14,304 reduced the current and constriction. B, similar recordings in a MV. UK 14,304
caused a constriction of the MV but had little effect on the oxidation current. The bars under the current traces in
A and B indicate the period of nerve stimulation (60 pulses with a 0.3 ms pulse width). C, frequency-dependent
inhibition of NA oxidation currents in MA and MV by UK 14,304. Data are expressed as a percentage of the
response obtained before UK 14,304. ∗ Significantly different from control. #Significantly different from MA. D,
frequency–response curves (1.0–20 Hz) for constriction in MA and MV in the absence (control) and presence of
UK 14,304. ∗ Significantly different from control for both MA and MV (P < 0.05). Data are means ± S.E.M.
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
830
J. Park and others
information from that of more traditional spillover
measurements. Amperometric recording and spillover
measurements are both sensitive to the fraction of released
NA that is transported away from varicosities. However,
with amperometric techniques, the distance from the
release site to the recording microelectrode is minimized
as are the number of variables that could alter NA
measurement. Spillover measurements require longer
stimulus trains to evoke detectable release and reflect an
NA released from large segments of blood vessels and many
nerve fibres.
Postjunctional differences in neuroeffector
transmission in MA and MV
Our observations that MV are more sensitive to the
constricting effects of sympathetic input than MA are
A
J Physiol 584.3
consistent with the literature (Kreulen, 1986; Hottenstein
& Kreulen, 1987; Luo et al. 2003). There are several
mechanisms that contribute to the differences in
neuroeffector transmission in MA and MV.
Firstly, the transmitters released from periarterial
and perivenous sympathetic nerves contribute to the
functional differences in MA and MV. The α 1 -adrenergic
receptor antagonist, prazosin, had a minor effect on neurogenic constriction of MA but it blocked MV constriction.
On the other hand, the P2X receptor antagonist, PPADS,
largely blocked constriction in MA but not in MV. These
observations are consistent with neurogenic constrictions
in MV being mediated by NA acting at α 1 -adrenergic
receptors, and by ATP acting at P2X1 purinergic receptors
in small MA, particularly at low stimulation frequencies
(Smyth et al. 2000; Galligan et al. 2001; Gitterman & Evans,
2001; Luo et al. 2003).
B
Artery
Vein
Diameter
20 μm
5s
5 pA
Current
5s
20 Hz
20 Hz
C
D
Artery/Cocaine (n = 7)
Vein/Cocaine (n = 8)
250
200
#
#
#
#
150
100
% Constriction
NE Overflow (% control)
300
Artery (n = 6)
Vein (n = 7)
Artery/Cocaine (n = 6)
Vein/Cocaine (n = 7)
40
30
#
20
10
#
50
0
0
1
3
7
10
20
Frequency (Hz)
1
10
Frequency (Hz)
Figure 8. Effect of cocaine on NA oxidation current and neurogenic constriction in of MA and MV
A, representative recordings of constriction (upper traces) and oxidation currents (lower traces) in a MA in the
absence and presence of cocaine (10 μM). Cocaine increased the current but had little effect on the constriction.
B, similar recordings in a MV. Cocaine did not change the NA oxidation current or constriction in MV. The bars
under the current traces in A and B indicate the period of nerve stimulation (60 pulses with a 0.3 ms pulse width).
C, frequency-dependent increase in the NA oxidation current in MA but not MV by cocaine. Data are of expressed
as a percentage of the response obtained before cocaine treatment in each tissue. #Significantly different from
control levels. D, frequency–response curves (1.0–20 Hz) for vasoconstriction in MA and MV in the absence (control)
and in the presence of cocaine. #Significantly different from control levels for both MA (P < 0.05). Cocaine did
not change constriction in MV at any stimulation frequency. Data are means ± S.E.M.
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
Neuroeffector transmission in arteries and veins
J Physiol 584.3
Second, there are kinetic differences in the neurogenic
constrictions of MA and MV. The kinetics of vascular
constriction are determined by the spatio-temporal
pattern of neurotransmitter release and clearance,
post-receptor signalling, density of innervation and the
width of neuromuscular cleft (Gonon et al. 1993; Kreulen,
2003b). The constriction rate is faster in MV than in
MA. The predominant constrictor in MA is ATP acting
at P2X1 receptors, which are ligand-gated cation channels
(North, 2000). The predominant constrictor in MV is NA
acting at α 1 -adrenergic receptors, which are G-protein
coupled receptors (Wier & Morgan, 2003). The faster rate
of venous constriction could be related to the different
signalling mechanisms used by P2X1 receptors that use
A
831
extracellular calcium to cause MA contraction (Gitterman
& Evans, 2001) and α 1 -adrenergic receptors that mobilize
intracellular calcium and alter the phosphorylation state
of contractile proteins (Kitazawa et al. 2000). The faster
rate of venous constriction might also involve selective
activation of postjunctional α 2 -adrenergic receptors in
MV. Selective expression of functional postjunctional
α 2 -adrenergic receptors in MV might also contribute to
the greater sensitivity of MV to the constrictor effects
of endogenously released NA. Using a microelectrode
calibration procedure that mimics neurogenic release,
we determined the apparent NA concentration caused
by the efflux from nearby varicosities. This permitted
comparison of endogenous NA concentration–response
B
Artery
Vein
Diameter
10 μm
10 pA
10 s
Current
3 Hz
3 Hz
C
D
500
#
400
#
#
300
#
#
200
Artery (n = 5)
Vein (n = 5)
Artery/Cocaine + Yohimbine (n = 5)
Vein/Cocaine + Yohimbine (n = 5)
40
Artery/Cocaine + Yohimbine (n = 7)
Vein/Cocaine + Yohimbine (n = 5)
% Constriction
NE Overflow (% control)
600
30
20
10
100
0
#
1
3
7
10
20
Frequency (Hz)
0
1
#
10
Frequency (Hz)
Figure 9. Effects of combined inhibition of NET and α2 autoreceptors on NA oxidation currents and
constrictions of MA and MV
A, representative recordings of constriction (upper traces) and current (lower traces) caused by a 3 Hz train of
stimulation of a MA in the absence and presence of cocaine (10 μM) and yohimbine (1 μM). B, representative
recordings of constriction (upper traces) and current (lower traces) caused by a 3 Hz train of stimulation in a MV
in the absence and presence of cocaine (10 μM) and yohimbine (1 μM). Combined drug application increased
the current response in MA and MV but the drugs increased constriction only in MA. C, frequency–response
curves (1–20 Hz, 60 pulses) for cocaine/yohimbine induced increases in NA oxidation currents in MA and MV.
Combined drug application increased NA oxidation currents in MA at low frequencies (< 7 Hz) but did not change
significantly the oxidation currents in MV. Data are expressed as a percentage increase in oxidation currents over
values obtained in the same blood vessels before drug application. #Significantly different from control values
(P < 0.05). D, frequency–response curves for neurogenic constriction of MA and MV in the absence and presence
of combined application of cocaine and yohimbine. Combined drug application increased MA constrictions at 1
and 3 Hz. #Significantly different from control values (P < 0.05). Constrictions of MV were not significantly affected
by combined drug application. Data are means ± S.E.M.
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
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J. Park and others
curves in the two vessel types. MA was less sensitive than
MV to the constrictor effects of endogenously released NA.
These data are consistent with previously published work
(Luo et al. 2003) showing that MV are more sensitive to
the constrictor effects of exogenous NA.
Prejunctional differences in neuroeffector
transmission in MA and MV
Frequency–response curves for the NA oxidation currents
measured at the surface of MV were left-shifted from those
for MA. Similar findings were obtained in NA spillover
studies from whole MA and MV segments, assessed
by high-performance liquid chromatography (HPLC)
(Smyth et al. 2000; Bobalova & Mutafova-Yambolieva,
2001). In both cases, the increased NA overflow
in MV relative to MA could be caused by several
factors.
Firstly, the mechanisms of NA release from perivenous
and periarterial sympathetic nerves could differ such that
individual action potentials release more NA in MV.
There are no data available to address this issue; however,
different populations of sympathetic nerves are known to
supply arteries and veins (Browning et al. 1999; Hsieh,
2000).
Secondly, there may be differential expression of
α 2 -autoreceptors by arterial and venous sympathetic
nerves. Prejunctional α 2 -adrenergic autoreceptors located
at the sympathetic nerve terminals are activated following
exocytotic release of NA and inhibit further release
(Langer, 1980). The absence or blockade of the
autoreceptor would lead to less regulated release and higher
junctional concentrations of NA. Blocking α 2 -adrenergic
receptors with yohimbine increased peak NA oxidation
currents in MA at all stimulation frequencies. The key
role prejunctional α 2 autoreceptors play in modulating NA
release from sympathetic nerves supplying rat mesenteric
arteries has been reported previously (Dunn et al. 1999).
Oxidation currents in MV were increased by yohimbine
only at low stimulation frequencies. Yohimbine also
increased the rate of rise of the NA oxidation current in MA
at 3 and 20 Hz in MA but it increased the rate of rise of the
oxidation current in MV at 3 Hz only. These data indicate
that NA release rate is tightly coupled to prejunctional
autoreceptors in MA but less so in MV. We also found
that UK 14,304 decreased NA oxidation currents in MA
and MV, but this effect was larger in MA. Taken together
these data indicate that prejunctional α 2 -adrenergic
autoreceptors play a more prominent role in regulating
NA release in MA than in MV. The reduced influence
of α 2 autoreceptors in MV may be due to differences in
autoreceptor density or spatial separation from release
sites.
Thirdly, differences in NA oxidation currents in
MA and MV might be due to differences in clearance
J Physiol 584.3
efficiency. Termination of the action of NA at the
neuroeffector junction depends on removal from
the neuroeffector junction by NET and low affinity
extraneuronal cation transporters, and by metabolism
and diffusion (Eisenhofer, 2001). Therefore, the junctional
concentration of NA and its postjunctional effect are
influenced by release dynamics, reuptake, metabolism
and diffusion (Esler et al. 1990). Most estimates indicate
that 90% of released NA is transported back into the nerve
terminal via NET so other mechanisms play only a minor
role in NA clearance. The NA that escapes recovery by
NET is what is detected by the microelectrode. Therefore,
the measured NA concentration is lower than the true
junctional concentration (Mermet et al. 1990; Gonon
et al. 1993; Dunn et al. 1999), but nonetheless reflects the
release and clearance processes. We found that inhibition
of NET by cocaine increased measured NA in MA
preparations. Similar results have been obtained
previously in studies using desmethylimipramine to
block NET in rat mesenteric arteries (Dunn et al. 1999).
In contrast, cocaine had relatively little effect on NA
oxidation currents in MV. These data indicate that NET
is an important determinant of NA concentrations in the
junctional cleft in MA while diffusion or metabolism are
more important for clearance in MV.
Combined application of cocaine and yohimbine
produced a modest increase in NA oxidation currents
in MV preparations without increasing constriction at
frequencies > 7 Hz. In some cases, constriction actually
decreased with the combined drugs. The unchanging
constriction maximum is likely to be due to saturation
of the postjunctional receptors on the vascular smooth
muscle cells due to the prolonged presence of high
junctional NA concentrations.
Summary and conclusions
The data presented herein indicate that the
time-dependent concentration of NA at artery and
vein surfaces, caused by complex mechanisms of
release and clearance at sympathetic varicosities, can
be studied in vitro using continuous amperometry.
The key finding from this work is that differential
neurogenic control of MA and MV was demonstrated.
These differences include, arrangement of perivascular
nerve fibres, neurotransmitters mediating constriction,
sensitivity to the constrictor effects of endogenous
NA, function of the α 2 -autoreceptor and NET. MA
are much less sensitive to the constrictor effects of
endogenously released NA than are MV. NA release and
clearance in the arterial neuroeffector junction is tightly
regulated by α 2 -adrenergic autoreceptors and NET.
While α 2 -adrenergic autoreceptors regulate NA release
at the perivenous neuroeffector junction, this effect is
C 2007 The Authors. Journal compilation C 2007 The Physiological Society
J Physiol 584.3
Neuroeffector transmission in arteries and veins
less prominent than in arteries. NA makes only a minor
contribution of neurogenic constriction of MA under our
experimental conditions even though it is released in both
MA and MV. NA, acting at prejunctional α 2 autoreceptors,
may function as a modulator of ATP release in small
MA. A similar conclusion has been reached by previous
investigators who studied sympathetic neuroeffector
transmission in guinea pig small intestinal submucosal
arterioles (Evans & Surprenant, 1992). NET contributes
little to NA clearance at the venous neuroeffector
junction. These differences in neuroeffector transmission
may contribute to the different haemodynamic functions
of arteries and veins. Rapidly developing and sustained
constrictions of veins are required to offset orthostatic
changes in blood distribution while phasic changes in
arterial tone would facilitate moment to moment blood
pressure control and organ perfusion.
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Acknowledgements
This research was supported by grants from the National
Institutes of Health, HL63973 and HL70687 (G.D.F. and J.J.G.),
and HL84258 (G.M.S.).
C 2007 The Authors. Journal compilation C 2007 The Physiological Society